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ATRAZINE
History:
Prior to the 1940s, the main method of weed control and field
clearance was manual labor, which was time consuming and expensive.
The first herbicide marketed was 2, 4-dichlorphenoxyacetic acid during
the
1940s, followed by other phenoxy acid compounds. Paraquat was initially
marketed in the early 1960s and was followed by the benzoic acid
compound dicamba later that decade. Since then there has been a
progressive increase in the use and development of herbicides
(Tominack,
2006).
Atrazine herbicides belong to the group of the most widely used
herbicides worldwide (Kang et al., 2003 and Prosen, 2012).
Development
of the ATZ began in the early 1950s to combat the weeds most plaguing
to
farmers (LeBaron et al., 2008).
These agrochemicals are used primarily as pre- and post-emergent
herbicides for the control of weeds in many agricultural crops like corn,
wheat, maize and barley. They interfere with the photosynthetic electron
transport chain in susceptible plants by binding to the quinine binding
protein in photosystem II (Strong et al., 2002).
Atrazine is one of the most commonly used triazine herbicides in
the world (Schuler et al., 2005) and frequently used in Egypt (Mekkawy
et al., 2013). ATZ toxicity occurs most frequently as an environmental
contaminant (Ackerman, 2007).
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Chemical properties:
Chemical Name: 2 – chloro – 4 - ethylamine-6-isopropylaminoStriazine,
some European countries have included ATZ on the list of
pesticide residues to be controlled because it is a potential contaminant
due to its chemical characteristics, including lipophilicity, slow
hydrolysis,
moderate to low water solubility, and high solubility in organic solvents
with high absorption by organic matter, clay, and fat tissues (Ross et al.,
2009).
Atrazine is prepared from cyanuric chloride, which is treated
sequentially with ethylamine and isopropyl amine. Like other triazine
herbicides, atrazine functions by binding to the plastoquinone-binding
protein in photosystem II, which animals lack. Plant death results from
starvation and oxidative damage caused by breakdown in the electron
transport process (Arnold et al., 2001).
Fig. (a): Chemical structure of atrazine (Kaune et al., 1998).
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Trade names: include Aatrex, Aktikon, Alazine, Atred, Atranex,
Atrataf, Atratol, Azinotox, Crisazina, Farmco Atrazine, G-30027,
Gesaprim, Giffex 4L, Malermais, Primatol, Simazat, and Zeapos (Stevens
and Sumner, 1992).
Physical properties (Kidd and James, 1991):
Appearance: Atrazine is a white, crystalline solid powder.
Molecular weight: 215.69.
Water solubility: 28 mg/L .
Solubility in other solvents: chloroform v.s.; diethyl ether v.s.;
dimethyl sulfoxide v.s.
Melting point: 176 C o.
Vapor pressure: 0.04 mPa .
Partition coefficient: 2.3404.
Adsorption coefficient: 100.
Atrazine biodegradation:
Atrazine remains in soil for months and can migrate from soil to
groundwater; once in groundwater, it degrades slowly in soil primarily by
the action of microbes. The half-life of ATZ in soil ranges from 13 to 261
days (Wackett et al., 2002).
Atrazine biodegradation can occur by two known pathways:
Hydrolysis of the C-Cl bond, followed by the ethyl and isopropyl groups,
catalyzed by the hydrolase enzymes called AtzA, AtzB, and AtzC. The
end
product of this process is cyanuric acid, itself unstable with respect to
ammonia and carbon dioxide. This pathway also occurs in Pseudomonas
species as well as a number of bacteria (Zeng et al., 2004).
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Toxicokinetics of atrazine:
(1) Absorption and distributions:
Atrazine is rapidly absorbed from the gastrointestinal tract, based on
tissue distribution in case reports of ATZ ingestion and on plasma
concentrations and urinary and fecal excretion in single dose studies in
rats
(Agopian et al., 2013).
About 60%-80% of an oral ATZ dose is absorbed in rats. The
absorption phase of ATZ compounds appears to be prolonged in humans
where the serum concentration continues to increase during treatment
with
hemodialysis (Brvar et al., 2008). It is readily absorbed through the
gastrointestinal tract. When a single dose of 0.53 mg ATZ was
administered to rats by gavage, 20% of the dose was excreted in the feces
within 72 hours. The other 80% was absorbed across the lining of the
gastrointestinal tract into the blood stream. After 72 hours, 65% was
eliminated in the urine and 15% was retained in body tissues, mainly in
the
liver, kidneys, and lungs (Tamura et al., 2001).
Dermal absorption of ATZ is incomplete but increases with exposure
to the proprietary formulation (McMullin et al., 2003). In vitro studies
using human skin, about 16% of the applied dose of ATZ was absorbed
by
the skin (Ademola et al., 1993).
Once absorbed by humans, ATZ is rapidly distributed and
metabolized with a biological elimination half life of 10.8 to 11.2 hours.
Some ATR and its metabolites may enter the organs or fat tissue (Agency
for Toxic Substances and Disease Registry, 2003).
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(2) Metabolism:
In vitro studies indicate that ATZ is metabolized primarily by
cytochromes P450 (CYPs) and, to a much lesser extent, by glutathione
transferases (Hanioka et al., 1999). The major in vitro detected CYPderived
metabolites of ATZ are the N-dealkylated products desethyl atrazine
(DEA)
and desisopropyl atrazine (DIA). However, didealkyl atrazine (DACT) is
the
major in vivo detected metabolite of ATZ in rat plasma (Brzezicki et al.,
2003) and in mouse plasma and urine (Ross and Filipov, 2006).
In humans, major ATZ metabolites detected in urine of
occupationally exposed subjects are DEA, DIA, and DACT (Catenacci et
al., 1993). Earlier studies reported that DEA and DIA were the two
primary
ATZ metabolites found in urine of occupationally exposed humans
(Ikonen
et al., 1988 and Catenacci et al., 1990). However, modern analytical
technology indicates that DACT is the most frequently detected human
urinary metabolite of ATZ (Barr et al., 2007 and Ross et al., 2009).
Fig. (b): Atrazine metabolism and major chlorinated metabolites (Fraites et al.,
2009).
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(3) Excretion:
The major route of excretion is urinary (EPA, 2002). Metabolites are
excreted in the urine and around 25% of them are conjugated to
glutathione
(McMullin et al., 2003), whereas only 2% of ATZ are excreted
unchanged
in urine (Perry et al., 2001 and Curwin et al., 2007). Fecal excretion of
ATZ and metabolites accounted for 14% of the dose in 24 hours and 19%
of the dose in 72 hours after dosing (Timchalk et al., 1990).
Toxicodynamics:
The primary target of ATZ in some animal species is the female
reproductive system. Altered estrus cyclicity has been observed in
SpragueDawley and Long-Evans. (Gojmerac et al., 1999).
A recent set of experiments has indicated that ATZ may disrupt
endocrine function, and the estrus cycle, primarily through its action on
the
central nervous system. In certain strains of rats, including SpragueDawley
and Long-Evans, reproductive senescence begins by one year of age, and
results from inadequate stimulation of the pituitary by the hypothalamus
to
release LH; low serum levels of LH leads to anovulation, persistent high
plasma levels of estrogen, and persistent estrus. Atrazine apparently
accelerates the process of reproductive senescence in these strains of rats
(Cooper et al., 2000).
Atrazine has been shown to induce mammary tumor formation in
female Sprague-Dawley rats, but not male Sprague-Dawley. This effect is
also thought to be the result of acceleration of reproductive senescence.
Both the failure to ovulate and the state of persistent estrus lead to
constant
elevated serum levels of endogenous estrogen, which may result in tumor
formation in estrogen-sensitive tissues. Therefore, the mechanism of
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disruption of normal reproductive cyclicity and mammary carcinogenicity
in these strains of rat likely does not involve direct interaction of ATZ
with
estrogen or the estrogen receptor (Stevens et al., 1999).
Sanderson et al. (2001) has demonstrated that ATZ and its two
primary metabolites, deethyl-and deisopropylatrazine, are capable of
inducing aromatase activity, with a corresponding increase in aromatase
ribonucleic acid (RNA), in the human adrenocortical carcinoma cell lines.
Aromatase is the rate-limiting enzyme in the conversion of androgens to
estrogens, and its induction could play a role in estrogen-mediated
pathologies.
Increasing the aromatisation of testosterone and conversion to
estrogen is further postulated that an increased estrogenic environment
may
favour i) induction of cancers and/or proliferation of pre-existing
estrogendependent
cancers and/or ii) altered relative sex hormone levels, which in
turn may have an adverse effect on a terminal or downstream end-point of
reproduction and/or development (Cooper et al., 2007).
Highly RISK GROUPS
A susceptible population will exhibit a different or enhanced
response to ATZ than other persons who exposed to the same level of
atrazine in the environment. Reasons may include genetic makeup, age,
health and nutritional status, and exposure to other toxic substances (e.g.,
cigarette smoke). These parameters result in reduced detoxification or
excretion of atrazine, or compromised function of organs affected by
atrazine. Atrazine has been shown to cause liver effects in animals;
therefore, people with liver damage or disease may be at greater risk from
exposure to atrazine (Aso et al., 2000).
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TOXICITY OF ATRAZINE
Acute toxicity:
Atrazine can be absorbed orally, dermally, and by inhalation.
Symptoms of poisoning include abdominal pain, diarrhea and vomiting,
eye
irritation, irritation of mucous membranes, and skin rashes. At very high
doses, rats showed excitation followed by depression, slowed breathing,
incoordination, muscle spasms, muscular weakness, hypoactivity,
hypothermia and convulsion (Californians for Alternatives to Toxics,
2009
and Jason, 2010).
Chronic toxicity:
Central nervous system:
Atrazine impaired motor coordination, movement and performance
(Podda, 2002).
Despite the fact that hormones shown to be affected by ATZ also
play a major role in the development of central nervous system. The
neurological effect of life time exposure to this herbicide may not
manifest
until late in life, such as impairment in movement and cognition
associated
with Parkinson's disease and dementia. Rodent models examining the
neurotoxic effect of ATZ indicate this herbicide produces dose-dependent
decreases in striatal dopamine level. Its well established that Parkinson's
disease is related to a reduction in striatal dopamine. Furthermore The
striatum is involved in cognition and attention processes that may be
affected in dementia. Therefore, it is possible that long term ATZ
exposure
could contribute to the development of parkinsonian symptoms or
cognitive
deficit associated with dementia (Agonli and Carli, 2011).
Immune system:
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Atrazine causes immune system failure in animals. This effect has
been shown in amphibians and laboratory rodents. In amphibians, ATZ
exposure impairs immune function and increases susceptibility to disease
(Cummings, 2001). Immune cells are unable to eliminate disease
pathogens and exposed amphibians are more likely to succumb to viral
diseases, bacterial infections and macroparasites, including the parasites
that cause limb deformities in amphibians (Kelce, 2001).
Similarly, ATZ exposure in rodents impairs immune function and
decreases an exposed animal’s ability to fight cancer and other diseases.
Further, its exposure in rodents can lead to hypersensitivity, making
exposed animals more susceptible to allergies. Most likely, the negative
effects on immune function are due to an ATZ-induced increase in the
stress hormones (corticoids). In salmon, it induced increase in stress
hormones in fresh water, impairs the ability of exposed fish to return to
the
ocean leading to high mortality in these commercially important fish.
(Gray et al., 2001).
Cardiovascular Effects:
Experimental studies showed that ATZ induced electro-cardiographic
changes consisted of slight to moderate increases in heart rate, moderate
decreases in PR values, slight decreases in QT values, atrial premature
complexes, and atrial fibrillation in both sexes, and degeneration of the
heart
muscles. Additionally, ATZ exposure leaded to enlargement and
softening
of the heart and thickened valves (Gammon et al., 2005 and Chan et al.,
2007).
Respiratory effects:
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No animal studies were evaluated respiratory function, mice gavaged
with a single dose of 875 mg/kg ATZ, sheep that consumed hay sprayed
with ATZ (approximately 47 mg /kg /day) for 25 days and pigs treated
with 2 mg/kg/day ATZ in the feed for 19 days had no gross or
histopathological lesions of the lungs (Ćurić et al., 1999).
Gastrointestinal effects:
No histological alterations were observed in the gastrointestinal
tracts of rats exposed to 52–71 mg/kg/day for 12–24 months or in sheep
exposed to approximately 47 mg atrazine/kg body weight/day for 25 days
(Johnson et al., 2002).
Hepatotoxicity:
In liver, organ responsible for detoxification process, with Wistar
rats orally exposed to 400 mg/kg body weight of ATZ for 14 days,
showed reduced accumulation of hepatic glycogen and early symptoms of
cytotoxicity. This event is attributed to the hepatotoxic effect of ATZ,
which inhibits the activity of key enzymes of glyconeogenesis such as
hexokinase, glycogen synthase, and glucokinase (Glusczak et al., 2006).
Another example of cellular biological process that could be changed in
response to ATZ exposure is the lipid metabolism and insulin resistance.
Study performed in Sprague- Dawley rats treated for 5 months with
vehicle or
ATZ (30 or 300 μg /kg /day ), supplied in drinking water, showed
prominent
accumulation of lipid droplets in the livers of ATZ treated rats. By means
of
transmission electron microscopy, some liver mitochondria from the
ATZtreated
group showed partially disrupted cristae (Lim et al., 2009).
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Long-term exposure to the herbicide ATZ might contribute to the
development of insulin resistance and obesity, particularly where a high
fat
diet is prevalent and cause damage to the liver and heart (Stevens and
Sumner, 1992).
Renal effects:
Kidney effects have been observed in rats and pigs, but not in mice,
sheep, or dogs. In male Wistar rats administered ATZ via gavage at 100
mg/kg/day or higher for 14 days, increases in urinary sodium, potassium,
chloride, and protein levels and serum lactate dehydrogenase and
γ-hydroxybutyrate dehydrogenase activities (Santa Maria et al., 2006).
Exposure of male rats to 52 mg/kg/day ATZ in the diet for 12
months resulted in decreased kidney weight, decreased specific gravity
and
increased volume of urine, and increased incidence of pelvic calculi in the
kidney; females exposed to 71 mg/kg/day had only increased relative
kidney weight . The rat data suggest that males may be more sensitive to
the renal toxicity of ATZ than females (Aso et al., 2000).
Subacute glomerulitis and degeneration and desquamation of the
proximal tubules were observed in female pigs receiving 2 mg/kg/day
atrazine in the diet for 19 days (Ćurić et al., 1999).
Hematological effects:
Although some animal studies have reported hematological effects,
the results have been inconsistent across studies. Decreases in
erythrocyte,
hemoglobin, and hematocrit levels and increases in mean platelet levels
were observed in female rats exposed to 71 mg/kg/day ATZ in the diet
for
12–24 months. (Dési, 2003).
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Musculoskeletal effects:
No histopathological changes were noted in skeletal muscle of male
or female rats. (Dési, 2003).
Teratogenic effects:
Atrazine does not appear to be teratogenic. (Draber, 1992).
Carcinogenic effects:
Atrazine did not cause tumors when mice were given oral doses of
21.5 mg/kg/day from age 1 to 4 weeks, followed by dietary doses of 82
mg/kg for an additional 17 months. However, mammary tumors were
observed in rats after life time administration of high doses of atrazine
(Kidd and James, 1991).
The carcinogenic potential of ATZ has been investigated in a number
of epidemiology studies, including cohort studies of workers at triazines
manufacturing facilities, case-control studies of farmers using ATZ or
triazines, and ecological studies of populations living in agricultural areas
with high ATZ use and residents living in areas with ATZ-contaminated
drinking water. In most of these studies, it is likely that the individuals
were exposed to ATZ via several exposure routes. For example, in the
studies of farmers, the likely exposure routes are inhalation during
application of atrazine, dermal during handling and use of ATZ, and
possible oral exposure due to contamination of groundwater (Hoar,
2002).
Epidemiological data are available for a number of types of cancers;
the most widely studied cancer type is non-Hodgkin’s lymphoma. In
general, case-control studies of farmers using ATZ (in some studies, data
are only available for triazine exposures) found small elevations in the
risk
of developing non Hodgkin’s lymphoma (Osburn, 2001).
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Evidence on the possible association between ATZ exposure and
increased risk of other cancer types is weak. Studies of farmers or
possible
agricultural workers did not find significant increases in the risk of
multiple
myeloma, leukemia, soft tissue sarcoma/carcinoma, or Hodgkin’s disease.
Suggestive evidence between atrazine (or triazines) exposure and an
increased risk of prostate cancer, breast cancer, and ovarian cancer have
been reported. Although these data provide a suspicion of
carcinogenicity,
the limited number of investigations and study limitations preclude
drawing
conclusions regarding these cancer types (Waring and Moore, 2000).
The animal data suggest that the carcinogenicity of ATZ is species-,
strain- and sex-specific. Statistically significant earlier onset of mammary
tumors or incidence of mammary tumors were observed in female
SpragueDawley rats, but not in female Fischer 344 rats. An increase in mammary
tumors was observed in male Fischer 344 rats; however, it is likely that
the
increased tumor incidence is due to increased life span of the ATZ-treated
animals, as compared to the controls (aged Fischer 344 rats have a high
rate
of spontaneous mammary tumors). The early onset of mammary tumors
in
female Sprague-Dawley rats is believed to be the result of ATZ-induced
acceleration of reproductive senescence. Both the failure to ovulate and
the
state of persistent estrus lead to constant elevated serum levels of
endogenous estrogen, which could result in tumor formation in
estrogensensitive
tissues (Portr, 1999).
IARC has classified ATZ as “not classifiable as to its carcinogenicity to
humans” (Group 3) based on inadequate evidence in humans and
sufficient
evidence in experimental animals (Osburn, 2001).
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Developmental effects:
Atrazine exposure has been associated with developmental effects in
both humans and animals. An association was found between Iowa
communities exposed to an average of 2.2 mg/L ATZ in the drinking
water in
1984–1990 and an increased risk of intrauterine growth retardation and
cardiac, urogenital, and limb reduction defects. The results of a survey of
farm
couples living year-round on farms in Ontario, Canada indicate that the
sex
ratio was not altered and the risk of small for gestational age deliveries
was
not increased in relation to atrazine exposure (Laws et al., 2000).
Developmental effects in response to oral exposure to ATZ have
been demonstrated in laboratory animals. Studies have shown that
gestational and pre pubertal exposure to ATZ has an effect on
reproductive
development in rats and rabbits. The effects of gestational exposure to
ATZ
in rats and rabbits include increased post-implantation losses, full-litter
resorptions, decreased live fetuses/litters, increased prenatal loss,
decreased
litter size, and reduced pup weights, which could be attributed to severe
maternal toxicity. Atrazine exposure in rats is also associated with
delayed
vaginal opening, first estrus cycle, and uterine growth for female rats and
decreased prostate weight, increased incidence and severity of
inflammation of the lateral prostate, increased myeloperoxidase levels in
the prostate, and increased total DNA in the prostate for male rats (Stoker
et al., 1999).
Atrazine has also been shown to have an effect on the development
of the nervous system in rats. Mild neurobehavioral effects were observed
in female offspring of Fischer rat dams exposed to ATZ pre-mating,
including increased spontaneous activity level, and male offspring had
improved performance (decreased latency and increased avoidance) in
avoidance conditioning trials (Ashby et al., 2002).
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Other developmental effects include incomplete ossification of the
skull, hyoid bone, teeth, forepaw metacarpals, and hind paw distal
phalanges in the offspring of exposed Sprague-Dawley rats, and non
ossification of metacarpals and middle phalanges, talus and middle
phalanges, and patella in the offspring of exposed rabbits. No
developmental effects were noted in a two-generation study in which rats
were exposed to ATZ in the diet (Narotsky et al., 2001).
Delayed onset of puberty occurred in young male and female rats
exposed to ATZ. Exposure to ATZ may be associated with mammary
tumors in at least one strain of adult rats (Stoker et al., 1999).
Effects reported in adults (human and experimental animals) include
shortening of estrous cycle length, attenuation of the LH (leutenizing
hormone) surge, decreases in pituitary hormone levels, ovarian
histopathology (changes in ovarian tissue), and liver effects including
increased serum lipids and liver enzymes, and liver histopathology. Other
effects on the central nervous system, immune system, and cardiovascular
function have been reported in adults. Exposure to atrazine may be
associated with some types of non-Hodgkin’s lymphoma in adult humans
(Cummings et al., 2000 ).
Atrazine toxicity in children:
There are very few studies of ATZ toxicity in children. One study
indicated that increased risk of preterm delivery and intrauterine growth
retardation correlated with increasing levels of ATZ in maternal drinking
water that contained a mixture of several pesticides . Decreased birth
weight was significantly associated with seasonal variations in ATZ
concentrations in drinking water. One study of childhood cancers (bone
and
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brain cancers, lymphomas and leukemias) found increased incidence of
these cancers was significantly associated with concentrations of three
chemicals (atrazine, nitrates, and metachlor) together in drinking water,
but
not any of the three chemicals alone. Significantly increased risk of
preterm
delivery, intrauterine growth retardation and decreased birth weight were
significantly associated with ATZ concentrations in drinking water
(Arbuckle et al., 2001).
Peruzović et al. (1995) found subtle neurobehavioral effects
(increased spontaneous activity in females and increased performance in
avoidance conditioning trials in males) in offspring of rat dams exposed
to
120 mg/kg ATZ 6 times during a 12-day period that ended 4 weeks
before
the rats were bred. The mechanism for this effect is unknown, but since
ATZ is not thought to persist in tissues, it may be mediated through
changes in the dam that later affect the offspring. These data indicate that
the developing organism may be susceptible to the effects of ATZ and/or
its metabolites. There are no studies that indicate that metabolism of ATZ
differs between children and adults or between young and adult animals.
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REPRODUCTIVE EFFECT:
Normal anatomy of the testis:
The testis is a paired, ovoid male reproductive organ that sits in the
scrotum, separated from its mate by a scrotal septum. Described by some
as
being shaped and sized like a large olive or small plum. The testis sits
obliquely with its long axis mostly vertical with a slight anterior and
lateral
slant to the superior pole. Superiorly, it is suspended by the spermatic
cord,
with the left testis often sitting lower than the right testis. Inferiorly, the
testis is anchored to the scrotum by the scrotal ligament, a remnant of the
gubernaculums (Sahni et al., 1996).
The tunica vaginalis testis (a remnant of the processus vaginalis)
envelopes the testis in a double layer, except at the superior and posterior
borders where the spermatic cord and epididymis adhere to the testes. The
visceral layer of the tunica vaginalis testis is closely applied to the testis,
epididymis, and ductus deferens. On the posterolateral surface of the
testis,
this layer invests a slit-like recess between the body of the epididymis and
the testis that is called the sinus of epididymis (Moore and Daley, 2006) .
The parietal layer of tunica vaginalis is adjacent to the internal
spermatic fascia, is more extensive, and extends superiorly into the distal
part of the spermatic cord. Deep to the tunica vaginalis, the tunica
albuginea is a tough, fibrous outer covering of the testis. On the posterior
surface, it is reflected inwardly to form an incomplete vertical septum
called the mediastinum testis (Sahni et al., 1996).
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Normal histology of the testis:
Fig.(c): Shows testis and epidydimal ducts (Junqueira and Carneiro, 2005).
Each testis is an ovoid, compact organ with a more or less crescent
shaped epididymis extending around its superior and poster lateral
borders.
Its outermost mesothelial covering represents the visceral layer of tunica
vaginalis, which is the membranous lining of a serous sac evaginated
from
the peritonium. Beneath the mesothelial covering is a thick capsule of
dense ordinary connective tissue that is known as the tunica albuginea
because of its whitish appearance. From this capsule, fibrous septa extend
inward and subdivides the interior of the testis into incomplete, roughly
pyramidal lobules (Kerr, 1991).
The septa converge toward the midline of the posterior border of the
testis, where they meet along a ridge like thickening of the tunica
albuginea
called the mediastinum testis, both ends of the component looped
seminiferous tubule open into a network of fine anastomosing channels
called rete testis. This network leads into a number of ductuliefferentes
(Kerr, 1991).
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Seminiferous tubules:
Seminiferous tubules are the main functional components of the
testis. Each of the several hundred seminiferous tubules in each testis is a
highly coiled tubule lined by a stratified germinal epithelium containing
various stages of spermatogenic cells. The seminiferous epithelium is
supported by the basement membrane (Trainer, 1987).
Sertoli cells:
Sertoli cells is the simple columnar supporting epithelium of
seminiferous tubules, they are nonproliferating cells. Wide gaps seems to
exist between these cells. Sertoli cells are characterized by a large pale
staining nucleus that generally lies towards the base of cell. Typically
elongated to ovoid sertoli cells possess an elaborate Golgi complex,
patches
of rough endoplasmic reticulum, and an extensive smooth endoplasmic
reticulum. Lipid droplets and crystalloid inclusions of unknown
significance are also present in the cytoplasm (Yeung, 1991).
Spermatogonia:
All regions of seminiferous tubules have a basally situated population
of spermatogonia. They are large and rounded cells lie adjacent to the
basement membrane of the tubule. They represent the dipliod cells from
which primary spermatocytes arise. Spermatogonia are a heterogenous
class
of cells made up of pale type A, dark type A, and type B spermatogonia.
The
two subtypes of type A spermatogonia are distinguishable by the depth of
staining of their respective nuclei (Cui et al., 2010).
The pale type A spermatogonia, relatively undifferintiated and with
extensive miotic potential, represent spermatogenic stem cells. Some of
their daughter cells remains undifferntiated as other pale type A
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spermatogonia. Such self renewal counteract depletion of the stem cell
population. The other daughter cells, roughly equal in number,
differentiate
into progenitors knows as type B spermatogonia that on dividing produce
primary spermatocyte (Cormack, 2001).
Spermatocytes and spermatid:
The large dividing cells in the middle third of the seminiferous
tubules are predominantly diploid primary spermatocytes. The resulting
spermatozoa are released into the lumen wall are haploid spermatids.
Some
spermatids have an elongated nucleus and are transforming into
spermatozoa which are released into the lumen of the tubule. The
sequence
of morohological changes that spermatids undergo when they transform
into spermatozoa is termed spermiogenesis (Cormack, 2001).
Spermatozoa:
Each spermatozoon consists of a head, midpeace (proximal portion
of the flagellum) and tail. The slightly flattened ellipsidal head contains
the
nucleus, which is densly packed with condensed chromatin. Anterioly,
the
nucleus is invested by the acrosomal head cap. The mid piece and the
reminder of the tail constitue the flagellum (Ross et al., 2002).
Interstitial (leydig) cells:
Distributes as scattered islands in the stromal loose connective tissue
between seminiferous tubules, they are the testosterone cells of the testis,
hence the name interstitial cells. Lying in close association with blood
capillaries or lymphatic capillaries, these steroid- producing cells are
fairly
large and have a more or less spherical nucleus. Their cytoplasm may
appear pale because of its substantial content of cholestrol- containg lipid
droplets. Crystalloid inclusions of unknows significance also chacterised
the cytoplasm (Cormack, 2001).
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Normal anatomy of ovary:
The ovary is a paired organ lie on the posterior wall of the pelvis
lateral to the uterus. They are supported by the suspensory ligaments, the
ovarian ligament, and the broad ligament, which also supports all of the
internal female genitalia. The ovary is a gland with a detached duct (the
fallopian tube), which "catches" the ovum as it is expelled from the
ovary.
The fallopian tubes are a pair of slender ducts through which ova pass
from
the ovaries to the uterus in the female reproductive system (Hansen et al.,
2008).
Normal histology of ovary:
The paired ovaries of the rat are grape-like structures that vary in
gross appearance and size, depending on the stage of the oestrous cycle.
Covering its surface is a single layer of modified peritoneal mesothelium,
the ovarian surface epithelium (OSE), which is continuous with the broad
ligament (mesovarium) that supports the ovary. The OSE of a single
ovary
can range from squamous to cuboidal, columnar or pseudostratified
columnar in type; this regional variation in OSE morphology
accompanies
the cyclical changes that occur within the underlying ovarian parenchyma
during the oestrous cycle (Kennedy and Mitra, 2003).
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Fig.(d):Subgross anatomy of the normal rodent ovary (mouse, H&E x400). The
cortex
(C) contains numerous follicles at various stages of maturation. The medulla
(M), which is not always present in histological sections, contains lymphatics,
nerves and numerous blood vessels (Long and Evans, 2002).
The ovarian stroma forms the body of the ovary and is composed of
spindle-shaped, fibroblast-like cells and delicate collagen fibres admixed
with ground substance. The stroma directly beneath the OSE is dense and
fibrous, and forms a narrow and variably distinct zone termed the tunica
albuginea. The ovarian stroma beneath the tunica albuginea is divided
into
a peripheral cortex and central medulla, although the latter is not always
visible in histological sections of ovary (Kerr, 1991).
The rete ovarii may be observed histologically within the rodent
ovary. This structure arises from cells of mesonephric origin which
migrate
into the developing gonad during embryogenesis. In the adult rat, the rete
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26
ovarii is composed of several groups of anastomosing tubules embedded
within the ovarian stroma and lined by a cuboidal or columnar epithelium
(Felicio et al., 2004).
In sexually mature rats, the cortex contains numerous follicles at
various stages of development. Five stages of follicular maturation
(folliculogenesis) are described:
Primordial follicle: This represents the earliest stage of follicular
development. Primordial follicles form during early foetal development
and
are typically located within the peripheral cortex, just beneath the tunica
albuginea. Each primordial follicle consists of a primary oocyte
surrounded
by a simple squamous follicular epithelium. Envelopment of the primary
oocyte by follicular cells arrests development of the germ cell at the first
meiotic division. During each oestrous cycle a cohort of “resting”
primordial follicles starts to develop into primary follicles; this process
occurs independently of hormonal stimulation up until the formation of
early tertiary follicles (Knobil et al., 2004).
Primary follicle: The squamous follicular cells surrounding the
primordial follicle differentiate into a single layer of columnar cells,
forming a primary follicle (Ross et al., 2002).
Secondary follicle: Proliferation of the columnar cell monolayer
results in the formation of a multilayered zone of granulosa cells, the
zonagranulosa, around the oocyte. This is accompanied by the
development
of a thick glycoprotein and acid proteoglycan coat, the zonapellucida,
between the oocyte and the zonagranulosa. As the secondary follicle
continues to grow, multiple fluid-filled spaces form within the
zonagranulosa; this stage is termed a vesicular follicle. Ovarian stromal
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27
cells surrounding the developing follicle become arranged into concentric
layers and form the theca folliculi, or theca. This layer is separated from
the
zonagranulosa by a basement membrane (Knobil et al., 2004).
Tertiary follicle: The cystic spaces within the zonagranulosa
coalesce and form a large central cavity, the follicular antrum. This cavity
is filled with fluid, the liquor folliculi, and surrounded by the
zonagranulosa. The primary oocyte is eccentrically positioned within the
tertiary follicle and resides within a mount of granulosa cells, called the
cumulus oophorus, that protrudes into the antrum. The granulosa cells
immediately surrounding the oocyte are termed the corona radiata.
The theca of the tertiary follicle is divisible into two zones: a theca
interna and theca externa. The theca interna consists of polygonal cells
with
vacuolated cytoplasm and open faced, vesicular nuclei. These cells
demonstrate the typical ultrastructural characteristics of steroid producing
cells (e.g. numerous cytoplasmic lipid droplets, large numbers of
mitochondria, and an extensive smooth endoplasmic reticulum), and are
the
main site of synthesis of androstenedione (a sex steroid intermediate). In
contrast, the cells of the theca externa are spindle-shaped and merge with
the surrounding ovarian stroma; they serve no endocrine function
(Hartman, 2000).
Preovulatory (Graafian) follicle: A small number of tertiary
follicles enter a preovulatory stage and undergo further morphological
changes. The follicular antrum continues to enlarge, causing attenuation
of
the surrounding zonagranulosa. Degeneration of the granulosa cells of the
cumulus oophorus occurs; this causes the primary oocyte to detach from
the zonagranulosa and float freely within the follicular antrum. The
primary
oocyte completes the first meiotic division just prior to ovulation and
forms
the secondary oocyte (Hubscher et al., 2005).
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28
Freely within the follicular antrum the primary oocyte completes the
first meiotic division just prior to ovulation and forms the secondary
oocyte
(Hubscher et al., 2005).
Following extrusion of the secondary oocyte from the Graafian
follicle, the granulosa and thecal cells of the follicle remnant undergo
hypertrophy and, to a lesser extent, hyperplasia. This process, termed
lutenisation, occurs under the influence of lutenising hormone (LH) and
prolactin, the two major luteotrophic hormones in rodents. Lutenisation is
accompanied by degeneration of the basement membrane separating the
theca interna and zonagranulosa, and infiltration of the postovulatory
follicle by blood vessels from the theca interna. The resulting mature
corpus luteum (“yellow body”) is a large eosinophilic structure that may
bulge out from the ovarian surface or obscure the ovarian
corticomedullary
junction, depending on its location (Knobil, 2004).
Fig. (e): Corpus luteum of rat ( H&E x400) (Knobil, 2004).
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29
The luteal cells (LC) comprising the corpus luteum are plump and
polygonal; they contain large nuclei and moderate amounts of
eosinophilic
cytoplasm. Cytoplasmic vacuoles form within luteal cells as the corpus
luteum matures and subsequently degenerates. Numerous blood vessels
(BV) are present, consistent with its function as a temporary endocrine
gland. Each corpus luteum matures during the oestrous cycle in which it
is
formed before regressing over the course of several subsequent cycles.
Consequently, at least three sets of corpora lutea are present within the
ovaries of normally cycling rats. Degenerating corpora lutea
progressively
shrink in size and are characterisedby increased amounts of fibrous tissue
and yellow-brown lipofuscin pigment. The fibrous tissue mass that
constitutes the corpus luteum during the final stages of regression is
termed
the corpus albicans (“white body”); this undergoes complete regression in
the rat, leaving no fibrous tissue remnant within the ovary (Long and
Evans, 2002).
Atrazine affects endocrine and reproductive systems. ATZ is thought
to bind to the androgen receptor, and it may affect the neuroendocrine
system by changing pituitary hormone levels such as leutenizing hormone
(LH) and follicle stimulating hormone (FSH) , both of which are critical
for
pregnancy (Andersen et al., 2002).
Atrazine exposure may affect germ cells (eggs and sperm). In one
study, reduced sperm number and motility were observed in male rats
injected with ATZ. In another study, decreased mating success
(pregnancy)
was observed following oral ATZ exposure of adult female (Haake,
2003).
Studies of embryotoxic effects following maternal oral or injection
exposure of rats to ATZ during pregnancy resulted in increased fetal
death;
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30
the doses that caused increased fetal death, as well as the degree of fetal
death at the higher doses, varied between genetically distinct strains of
rats.
In another study, prenatal exposure of rats and rabbits via oral maternal
exposure to ATZ resulted in embryotoxic effects only at doses that
caused
severe maternal toxicity (Snick et al., 1997).
Delayed mammary gland development in female offspring at puberty
was reported following exposure of female rats in utero and during
lactation via gavage (tube-feeding) of their mothers. Exposure in utero or
during lactation each alone resulted in delayed mammary gland
development in female offspring (Andersen et al., 2002).
Onset of puberty in male rats was delayed following prepubertal oral
exposure to ATZ or ATZ metabolites. In these studies, ATZ exposure
resulted in decreased food consumption and weight loss, and the authors
concluded that decreased food consumption contributed to some or all of
the observed effects of delayed onset of puberty in these specific studies.
No such delays in the onset of puberty were observed in a separate study
in
rats (Stoker et al., 2001).
Onset of puberty in female rats was also delayed following
prepubertal oral exposure to ATZ or ATZ metabolites. These delays were
attributed to ATZ exposure and not to decreased food consumption (Gray
et al., 1999).
Atrazine induced male reproductive toxicity:
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31
Kniewald et al. (2000) reported significantly decreased body weight
and relative weight of ventral prostrate, testis, sperm motility, sperm
number in epididymis. The histopathology of testis revealed cell
disorganization, cell cluster together with spermatocytes and various
degenerative changes. Electron microscopy evaluation of testis revealed
vacuolated cytoplasm, reduced collagen fibers, irregular shaped leydig
cell
degenerative changes in sertoli cells when ATZ was administered intra
peritoneally at the dose rate of 60 and 120 mg/kg bw twice a week over
60 days in rat.
The onset of puberty in the male rat involves a complex interplay of
several hormones including LH, FSH, testosterone and prolactin, prior to
the onset of puberty. LH stimulates testosterone secretion by the Leydig
cells. At the same time, LH secretion varies only slightly as puberty
approaches. However, there is an increased sensitivity of the testes to LH
prior to puberty, due to other hormonal influences, such as increased
prolactin secretion, that facilitate an up regulation of LH receptors (Peters
and Cook, 1973).
In contrast, there is a higher threshold for the gonadotropin/ gonadal
steroid feedback mechanism in the adult male as compared to the
immature male, making the immature male more sensitive to the
feedback
of testosterone. As this feedback sensitivity decreases, the
hypothalamicpituitary
unit becomes more effective at stimulating testicular
development, because there is less inhibition of gonadotropins by
testosterone (Šimić et al., 1994).
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32
Development of the size of the penis and cornification of the
epithelium of the prepuce and preputial separation in immature rats are
regulated by androgens (Marshall, 1999).
A decrease in testosterone during the juvenile period can delay
preputial separation and reduce the size of the androgen-dependent
tissues,
such as the ventral prostate and seminal vesicles. Normally, testosterone
levels rise gradually from PND 20 to 40, and abruptly double by PND 50
Atrazine exposure has been shown to alter LH and prolactin secretion in
female rats. An effect on LH and prolactin secretion in immature male
rats, and thus on pubertal onset, may also be possible (Matsumoto and
Monosson, 1999).
Atrazine induced female reproductive toxicity:
The onset of puberty in the female is a transitional period that
culminates with the initiation of cyclic surges of luteinizing hormone
(LH)
from the pituitary that stimulate ovulation. Vaginal opening generally
coincides with the first ovulation and occurs at 32 or 33 days of age in the
female rat. The hormonal changes which induce the first ovulation are
similar in many respects to the hormonal changes which induce all other
ovulations in rodents. The sequence of hormonal changes preceding the
first ovulation is as follows:
1. Serum estradiol levels increase followed by;
2. A dramatic increase (surge) in serum luteinizing hormone(LH);
3. Serum prolactin levels dramatically increase concomitant with the LH
surge (Kniewald et al., 2007).
Exposure to ATZ has been shown to attenuate the proestrus LH and
prolactin surges in Long-Evans and Sprague-Dawley rats. Since both of
these hormones are important for normal pubertal development, it is
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33
reasonable to hypothesize that atrazine may affect the onset of puberty in
the female rodent. In addition, reports that ATZ can reduce hypothalamic
norepinephrine concentrations and that intravenous injections of GnRH
restore the estrogen-induced secretion of LH in ovariectomized,
atrazinetreated
female rats (Cooper et al., 2000).
Wetzel et al. (1994) studied that lengthening of the estrous cycle,
increased number of days in estrus or under the influence of exposure to
estrogen, early onset of galactocele formation, early onset of mammary
and
pituitary tumor formation, and an increased incidence of mammary and
pituitary tumors when dietary administration of ATZ was made to Fischer
344 and Sprague-Dawley female rats.
Chlorinated metabolites of ATZ (e.g., diethylatrazine (DEA),
diisopropylatrazine (DIA), and diaminochlorotriazine (DACT)) are
considered equivalent in toxicity to ATZ, and exposure to metabolites are
also of concern (Wackett et al., 2002).